1. Field of the Invention
The invention relates to a method for producing glucosamine by culturing microorganism with a novel low-cost medium.
2. Description of the Prior Art
Glucosamine is one of the constitutional ingredients for articular cartilage, which can provide nutrition for articular tissue, enhance the ability of synovial fluid for recovering lubricating function, promote the regeneration of retrograded joints, and hence effectively reduce the pain generated from bone friction, as well as prevent the aggravation of arthritis condition. Glucosamine can be synthesized by human body itself. However, as age increased, the synthesis speed for glucosamine in the human body is slower than the decomposition speed of glucosamine, and consequently, the body and joints tend to be in short of glucosamine, and further, the metabolism of cells in joints may be affected.
The features of glucosamine include (1) stimulating the regeneration of chondroblast, promoting metabolism thereof, supplying nutrition for bone, reducing inflammation, and vanishing paint; (2) protecting cartilage cells from damage by drugs and external force, and preventing degeneration of joint; (3) increasing the amount of synovial fluid and viscosity thereof, enhancing the lubrication effect of the joint, and improving the function of the joint; (4) ameliorating sore waist and backache. In Europe, glucosamine has been used widely in treating osteo-arthritis. Once administrated, glucosamine can be absorbed fast, delivered to and used by various tissues in the body. The rate acute toxicity assay and microorganism mutagenicity study had shown that glucosamine is a safe and nontoxic health product, and supplement of glucosamine can prevent arthritis.
Glucosamine may be obtained naturally by extracting from chitin in carapace of marine shrimp and crab, as well as artificially by chemical synthesis. At present, industrial production of glucosamine is still carried out by hydrolyzing the carapace of shrimp and crab in hydrochloric acid solution. A conventional method for producing glucosamine comprises of hydrolyzing chitin with acid or enzymes. However, shrimp and crab carapaces obtained from different sources may affect the purity of glucosamine. In addition, glucosamine produced form contaminated shrimp and crab carapaces may be toxic. Furthermore, before hydrolyzing the shrimp and crab carapaces, rinsing of these carapaces is necessary and takes a lot of time and work to prevent notorious stinks. Moreover, glucosamine is not the only one product produced during the process of hydrolyzing the carapaces. Additional purification steps are required in order to isolate glucosamine and other by-products. In the consideration of reducing those tedious pre-treatment steps and diminishing allergic side effect sequela after taking up in the human body, this invention adopts microorganism producing approach instead of a conventional chemical method.
In addition to the above-described hydrolysis method, two methods for producing glucosamine from microorganism are currently used. One of the methods consists of decomposing chitin by means of intracellular and extracellular enzymes of fungus, while the other method consists of converting primary metabolism medium for microorganism into the secondary metabolism medium, for producing glucosamine from the medium.
The study of Deng et al in 2005 pointed out that currently, gene transfer technique had been used to produce glucosamine-producing Escherichia coli. Unfortunately, since the regulatory mechanism involved was too complicated, the detectable amount of glucosamine in the medium for E. coli became extremely low, only several milligram per liter. E. coli produced by gene transfer technology could increase the yield of amino-sugars. Said gene transfer strategy involved the promotion of genes associated with the function and catabolism of glucosamine, as well as the overexpression of the glucosamine synthase gene. Said method could increase up to 15-times of the glucosamine yield, but its titer still remained at the level of milligram. Feedback inhibition of glucosamine synthase had been identified to be a critical factor for applying said method. Further, screening of enzymes might increase the production of glucosamine, and could increase its titer to the level of several grams. Unfortunately, fast degradation of glucosamine in host cell, inhibition effect of glucosamine and degradation product thereof might hinder the increase of the glucosamine concentration [Deng, Ming-de, K. D. Severson, D. A. Grund, S. L. Wassink, R. P. Burlingame, A. Berry, J. A. Running, C. A. Kunesh, L. Song, T. A. Jerrell and R. A. Rosson, From Conceptto Process: Metabolic Engineering for Production of Glucosamine and N-Acetylglucosamine, Metabolic Engineering, 7(3), 201-214 (2005)].
In conventional technique and literature, study aimed at producing glucosamine by a way of secondary metabolites of microorganism is neither much nor comprehensive, and only following few fungi have been mentioned to contain glucosamine in their secondary metabolites:
1. Monasus: Most of the attention had been focused on contents monacolin K and GABA, far less on the fact that Monasus contained glucosamine. Hsieh et al. pointed out in their study in 2007 that in a medium consisting of 20 g/L rice bran, 25 g/L B-grade white crystal sugar, 15 g/L ammonium chloride, Monascus pilosus could produce 0.72 g/dm3 of glucosamine, with its optimal condition as: pH 5, and 30° C. [Hsieh, J. W., H. S. Wu, Y. H. Wei, and S. S. Wang, Determination and kinetics of producing glucosamine using fungi, Biotechnol. Prog., 23, 1009-1016(2007)].
2. Aspergillus: Aspergillus is a filamentous fungus widely present in nature world. Many Aspergillus fungi may produce secondary metabolites harmful to human body. Among them, the most well-known one is aflatoxin produced by Aspergillus flavus and Aspergillus parasiticus. Aflatoxin had been identified as a carcinogenic substance. Nevertheless, Hsieh et al. had indicated in their study in 2007 that, in a certain medium, Aspergillus sp. could produce 3.43 g/dm3 of glucosamine, with optimal condition as: pH 7 and 30° C. [Hsieh, J. W., H. S. Wu, Y. H. Wei, and S. S. Wang, Determination and kinetics of producing glucosamine using fungi, Biotechnol. Prog., 23, 1009-1016(2007)].
In view of the foregoing, conventional methods for producing glucosamine still have many disadvantages, and among the other, the production of glucosamine from microorganism fails to increase greatly as well as the cost of the medium is impossible to cut down. Consequently, conventional methods are not well-designed and require further improvement.
The inventors had learned various disadvantages derived from the above-described conventional methods for producing glucosamine, and devoted to improve and innovate, and finally, after studying intensively for many years, has developed successfully a method for producing glucosamine by culturing microorganism with a low-cost medium.